Immuno polymerase chain reaction assay

ABSTRACT

A method to detect biological molecules in a sample which is an immuno polymerase chain reaction assay which comprises the detection of a complex which includes a ligand: nucleic acid conjugate bound to at least one biological molecule. The complex is detected by the addition of a second nucleic acid molecule which is adapted to anneal to the nucleic acid of the conjugate.

The invention relates to an immunoassay which utilises the polymerasechain reaction.

The use of antibodies as a diagnostic tool is well documented, seeDiagnostic Applications of Monoclonal Antibodies to Human Cancer, ArchPathol Lab Med 108, 2, 101-105 (1985); Monoclonal Antibodies andColorectal Carcimoma: a clinical Review of Diagnostic Applications 12,3, 314-323 (1994); Diagnostic Approach to Phospholipid-DependentAntibodies: State of the Art Lecture Haemostasis, 29, 2-3, 135-149.Monoclonal antibodies, in particular, are advantageous since they arehighly specific for epitopes found in antigens which means a high degreeof specificity can be obtained when testing for antigens which arespecifically expressed in disease tissue providing a reliable measure ofthe expression of the antigen and accurate diagnosis of a disease whichis correlated with expression of the antigen (i.e. tumour rejectionantigens in cancer). It is also well documented that the detection ofnucleic acid by, for example hybridisation or the polymerase chainreaction (PCR), using nucleic acid probes has also been used in thediagnosis of disease. DNA probes can be relatively easily andinexpensively synthesized using oligonucleotide synthesis. Thespecificity of a nucleic acid probe is determined by its sequence andhow homologous the sequence is to the nucleic acid which is to bedetected. However a problem associated with both antibodies and nucleicacid probes is a degree of non-specific binding of the antibody ornucleic acid probe to assay products and/or other proteins and nucleicacid. A diagnostic test has to have a very high degree of reliability ifit is to have value in predicting the early on set of disease.

There are a number of molecules present within serum, for example,interleukins and parathyroid hormone related protein, which arepotential markers of cancer and other pathological conditions. Currentlythese are only measurable during the late stages of the disease processwhen they are overexpressed by tumours. Under normal conditions theproteins are present at concentrations <0.1 pM. Moreover, the earlydetection of pathogenic organisms in an infection can be critical towhether or not an infected animal survives the infection This isparticularly the case in diseases such as bacterial meningitis andsepticemia caused, for example by Staphyloccocus aureaus. The earlierthese molecules can be measured during the disease process the betterthe prognosis. However, early detection means that the molecules are atlow concentrations and the signaling/quantitation systems of currentimmunoassays, using enzymes and chemiluminescence does not providesufficient sensitivity to measure at these low levels.

In addition, there are many clinical scenarios where the simultaneousmeasurement of more than one analyte in a sample would be of significantdiagnostic and therapeutic benefit. Some obvious examples relate to theinvestigation of endocrine abnormalities where the interpretation of asingle result would be significantly affected by another result.

In thyroid disease a combination of thyroid stimulating hormone (TSH)with an index of thyroid hormone status (total or free thyroid hormone)is essential to make an accurate diagnosis in many cases. Ininvestigation of calcium disorders a combination of Parathyroid Hormone(PTH) and Parathyroid Hormone Related Protein (PTHrP) woulddifferentiate the two major causes of hypercalcaemia and elucidate thepresence of dual pathology which is often overlooked. When problems ofsexual dysfunction and infertility are investigated the pituitaryhormones luteinising hormone (LH) and follicle stimulating hormone (FSH)are measured with oestradiol and/or testosterone included in the hormoneprofile. Another major diagnostic area is the screening for Down'ssyndrome where a combination of hormone measurements (Human ChorionicGonadotrophin (HCG), alpha feto-protein (αFP)) and maternal age is usedto assess risk of the fetus having Down's syndrome. It is clear that thedevelopment of an assay system able to simultaneously measure a numberof analytes would have numerous diagnostic applications.

Sano et al (Science (1992), 258:120-122) has developed a technique inwhich a biotinylated double stranded DNA (dsDNA) template is bound to abiotinylated detector antibody during the final stages of an immunoassayusing streptavidin. PCR is undertaken to amplify the dsDNA, quantitationbeing achieved through gel electrophoresis and scanning equipment. Thetechnique is known as “immuno-PCR”

Immuno-PCR is a method which combines both antibody technology and thepolymerase chain reaction or other means to detect a nucleic acid probeconjugated to the antibody. In essence, immuno-PCR utilises an antibodyto which a nucleic acid probe has been conjugated. The conjugate bindsan antigen to be detected via the antibody part and non-bound conjugateis washed from the sample. The bound antibody is then detected by a PCRreaction which amplifies the nucleic acid part of the conjugate. Theassay provides a sensitive and specific test for a biological moleculewhich benefits from both the advantages discussed above. Specificity isprovided by the antibody and sensistivity by the PCR detection of thenucleic acid conjugated to the antibody.

In WO94/26932 an immuno-PCR method is disclosed which is referred to asNucleic Acid Tagged Immunoassay or NATIA which involves theimmobilisation of an antigen or antibody to a solid support. In thisexample either the antibody is conjugated to an oligonucleotide which isthen use to detect the immobilised antigen or the antibody isimmobilised and the antigen conjugated to the oligonucleotide. In eithercase the bound antibody/antigen is detected indirectly by PCRamplification.

In WO9632640 a variation on immuno-PCR is disclosed. The assay describedutilises an RNA dependent RNA polymerase. The conjugate comprises anantibody and a DNA template for an RNA transcript. The bound conjugateis detected by transcribing the DNA template into RNA using an RNAdependent RNA polymerase, such as QB replicase. The enzyme is able totranscribe RNA from the DNA template but with lower efficiency than aRNA template. Methods to conjugate nucleic acid to protein molecules areknown in the art. For example U.S. Pat. No. 5,635,602 disclosesantibody/DNA conjugates and methods for making same.

These techniques demonstrate increased sensitivity. However there aredisadvantages associated with the prior art techniques. For example,background resulting from non-specific binding via the nucleic acidmoiety or the antibody to plastic of the assay wells is problematic.Moreover the quantitation sytems are insensitive. In order to addressthese issues we have developed an alternative approach termed MultipleAnalyte Quantitation through Single Stranded Extension (MAQSSE).

An oligonucleotide is conjugated to a ligand which has specificity for abiological molecule. The ligand/DNA conjugate is incubated with a sampleand binds a target biological molecule. A single stranded DNA template(ssDNA) of defined length is then added to the reaction and anneals tothe bound oligonucleotide. A DNA polymerase and deoxynucleotidetriphosphates are added and the reaction heated to to elongate theprimed oligonucleotide strand to produce a dsDNA. A nuclease specificfor ssDNA is added to the reaction to degrade the background ssDNAtemplate, resulting in no detectable background template. To generate ameasureable signal, PCR is undertaken to amplify the double stranded DNAfollowed by detection using conventional techniques.

A variation of the above method is also disclosed which greatlysimplifies the assay and removes the need to add an exogenous singlestranded nuclease to remove the ssDNA template remaining in the reactionmix. The variation comprises a ligand:oligonucleotide conjugate whereinthe oligonucleotide has a bipartite sequence structure, (illustrated inFIG. 1 as “a,” and “b”). The conjugate thus formed is contacted with atest sample which potentially includes biological molecule to which theligand binds.

The bound conjugate is then incubated with the ssDNA template. Thebipartite oligonucleotide is complementary over part of its length to aregion of the ssDNA. The annealed bipartite oligonucleotide is extendedby DNA polymerase to form a double standed DNA. An excess ofoligonucleotide primer is added to the reaction mix, the sequence ofwhich is complementary to that part of the bipartite oligonucleotidewhich is not annealed to the ssDNA template. A polymerase chain reactionis then conducted. Only the ssDNA which has annealed to the bipartiteoligonucleotide is capable of being subsequently amplified therefore theassay provides a highly specific and sensitive means to monitor thepresence of biological molecules.

According to an aspect of the invention there is provided a method todetect at least one biological molecule comprising providing means todetect said biological molecule which means comprises a ligand:nucleicacid conjugate wherein the binding of the conjugate to said biologicalmolecule is detected by a polymerase chain reaction which detects asecond nucleic acid molecule which is adapted to anneal to the nucleicacid of the conjugate.

According to a further aspect of the invention there is provided amethod to detect at least one biological molecule wherein said methodcomprises the steps of:

i) providing a preparation comprising;

-   -   a) an assay sample to be tested; and    -   b) a ligand which is coupled to an oligonucleotide wherein said        ligand can bind at least one biological molecule in said sample;

ii) incubating said preparation under conditions which allow the bindingof said ligand to said biological molecule to form a complex;

iii) contacting the complex formed in (ii) with a single strandednucleic acid molecule adapted to anneal to at least part of theoligonucleotide coupled to said ligand;

iv) providing a polymerase which is capable of elongating theoligonucleotide annealed to said single stranded nucleic acid moleculeto form a double stranded nucleic acid;

v) incubating the preparation formed in (iii) with a nuclease whichdegrades the unannealed single stranded nucleic acid molecule;

vi) providing a polymerase and reaction conditions which amplify thedouble stranded nucleic acid molecule in (iv); and optionally

vii) detecting the presence of the amplified product formed in (vi).

In a preferred method of the invention there is provided an assay sampleselected from the group consisting of a sample of: blood; serum; semen;lymph fluid; cerebrospinal fluid; tears; saliva; urine; sweat.

In a further preferred method of the invention said ligand is apolypeptide.

In a yet further preferred method of the invention said polypeptide isan antibody, or at least the effective binding part thereof. Preferablysaid antibody is a monoclonal antibody, or at least the Fab fragment ofsaid monoclonal antibody.

In a further preferred method of the invention said biological moleculeis associated with a disease condition, for example cancer e.g. a tumourrejection antigen. Tumour rejection antigens are known in the art, forexample and not by way of limitation, the MAGE, BAGE, GAGE and DAGEfamilies of tumour rejection antigens, see Schulz et al Proc Natl AcadSci USA, 1991, 88, pp 991-993. Other examples include hormones e.g.thyroid stimulating hormone.

In a further preferred method of the invention said biological moleculeis a polypeptide, preferably an antigenic polypeptide expressed by apathogen. For example a viral, bacterial or parasitic pathogen.

In a further preferred method of the invention said polypeptide is areceptor. Alternatively said polypeptide is a ligand for a receptor.

It will be clear to someone skilled in the art that the ligand can be anantibody which is specific for a biological molecule which may bepresent in said assay sample. Alternatively the biological molecule maybe labelled with the oligonucleotide and the antibody specific for saidbiological molecule detected in the assay sample.

In a further preferred method of the invention said polymerase is a DNApolymerase. Preferably said DNA polymerase is selected from the groupconsisting of: E.coli DNA polymerase I; large fragment of E.coli DNApolymerase I, also referred to as Klenow fragment; T4 and T7bacteriophage DNA polymerase; modified T7 bacteriophage polymerase(referred to as Sequenase™).

Thermostable DNA polymerases are also included although these enzymesonly have optimal activity at 70-80° C. Thermostable DNA polymeraseswill have reduced activity at the reaction temperatures used in themethod according to the invention but they nevertheless have activity.Examples of thermostable DNA polymerases are so called Taq polymeraseisolated from the thermophilic bacterium, Thermus aquaticus. Otherexamples include thermostable DNA polymerases isolated from Thermusthermophilus; Thermosipho africanus; Thermotosa maritima.

In a preferred embodiment of the invention said DNA polymerase is T4bacteriophage DNA polymerase.

In a further preferred embodiment of the invention said nuclease is asingle stranded nuclease. Preferably said single stranded nuclease is S1nuclease or mung bean nuclease.

In a further preferred embodiment of the invention the polymerase usedto amplify said double stranded nucleic acid is a thermostable DNApolymerase. Preferably a thermostable DNA polymerase as hereinbeforedescribed.

The amplified product may be analysed by means known in the art whichallow the detection and/or quantitation of the DNA product. Typicallythis includes spectroscopy; fluorimetry, gel electrophoresis (agarose,polyacrylamide).

According to a further aspect of the invention there is provided aligand:oligonucleotide conjugate wherein said oligonucleotide isadapted, over at least part of its length, to anneal to a singlestranded nucleic acid by complementary base pairing.

According to a further aspect of the invention there is provided aconjugate according to any previous aspect of the invention whichfurther comprises an annealed single stranded nucleic acid molecule.

According to a yet further aspect of the invention there is provided aconjugate according to any previous aspect of the invention whichcomprises an annealed single stranded nucleic acid molecule wherein saidconjugate is bound to the ligand binding domain of at least onebiological molecule.

In a preferred embodiment of the invention said oligonucleotide is atleast 10 base pairs long. Preferably, at least 20 base pairs long. Morepreferably still said oligonucleotide is between 10-50 base pairs long.

Preferably said second nucleic acid is a single stranded DNA.

In a further preferred embodiment of the invention said ligand is apolypeptide. Preferably said ligand is an antibody, or the effectivebinding part thereof. Preferably said antibody is a monoclonal antibody.

According to a further aspect of the invention there is provided amethod to detect at least one biological molecule comprising the stepsof:

i) providing a preparation comprising;

-   -   a) an assay sample to be tested; and    -   b) a conjugate according to the invention;

ii) incubating said preparation under conditions which allow the bindingof said conjugate to said biological molecule to form a complex;

iii) contacting the complex formed in (ii) with a single strandednucleic acid molecule adapted to anneal to a part of the oligonucleotidein said conjugate;

iv) providing a polymerase which is capable of elongating and amplifyingthe annealed oligonucleotide and at least one oligonucleotide primerwherein said primer comprises a sequence complementary to that part ofthe oligonucleotide to which said single stranded nucleic acid is notannealed; and optionally

v) detecting the presence of the amplified product formed in (iv).

In a preferred method of the invention said oligonucleotide primer is apalindromic sequence.

A palindromic sequence is a sequence which has the same sequence whenread in a 5′-3′ direction as when read in a 3′-5′ direction. For examplea palindrome of the sequence: 5′ GGGCAAACGGG 3′ is 3′ GGGCAAACGGG 5′.

The use of a single palindromic primer to amplify the ssDNA allowsaccurate PCR conditions to be established thereby providing an reliabletest.

In a preferred method of the invention said method detects two or morebiological molecules.

In a further preferred method of the invention said method detects aplurality of biological agents.

An embodiment of the invention will know be described by example onlyand with reference to the following figures:

FIG. 1 shows a schematic diagram of an embodiment of the presentinvention;

FIG. 2 is an agarose gel electrophoresis of a thyroid stimulatinghormone (TSH):oligonucleotide conjugate;

FIG. 3 is an analysis of partially purified conjugate through a PD10column;

FIG. 4 is PCR amplification of conjugate aliquots from column fractions;

FIG. 5 is a standard curve for TSH detection using a 1:1000 dilution ofconjugate; and

FIG. 6 is a standard curve for TSH detection using a 1:5000 and a1:25000 dilution of conjugate;

MATERIALS AND METHODS Oligonucleotide & Template Design

A 100 base single strand oligonucleotide (GAT TTA ATC TGT ATC AGG CGGGTA TGG AGT ATA ATC TAG TAG AGA GTT AAGTAT GTA ATA TCG TTA AGC TAA TCTTAT GGA TAA AAA TGC TAT GGC AT ssTemp) was designed using Oligo6software in conjunction with database searches, together with comboC(GAT TTA ATC TGT ATC AGG CAT GCC ATA GCA TTT TTA TC) and revC (GAT TTAATC TGT ATC AGG C) oligonucleotides. The revC oligonucleotide wasdesigned to anneal to the 3′ terminal of the ssTemp, the comboC isdesigned to anneal to the 5′ terminal of the ssTemp, thisoligonucleotide also contains the revC sequence at its 5′ terminal.ssTemp exhibits no significant homology with human nucleic acidsequences and contains no internal annealing sites for either comboC orrevC. These oligonucleotides were commercially synthesized.

Conjugation

500 ul of 1 mg/ml anti human TSH was desalted using a PD10 column andresuspended in 100 mM sod phos 5 mM EDTA pH6.0. Fractions containing theantibody were pooled to a volumne of 1 ml and added to 1 vial 2mercaptoethlyamine (2-MEA) and incubated at 37′ C. for 90 mins. Thesolution was applied to two PD10 columns, 500 ul to eachpre-equilibrated with PBS 5 mM EDTA pH7.15. 1 ml of antibody containingfractions was collected and pooled.

200 ul of amino-labeled comboC oligonucleotide 2 mg/ml in water) wasadded to 250 ul of water followed by 50 ul of 1M sodium phosphate pH8.0.3 mg of succinimid 4 (N-maleimidomethyl)cyclohexane-1-carboxlate (SMCC)added, vortexed and the solution heated to 37′ C. for 20 mins. Thesolution was applied to a PD10 column pre-equilibrated with PBS pH7.15.Oligonucleotide containing fractions were identified using Oligreen(Molecular Probes)and pooled (1.5 ml). 1 ml of reduced anti TSH antibodyand 1.5 ml SSMCC derived amino comboC oligonucleotide were pooled andconcentrated to 100 ul using a Sartorius 5 Kda spin column.

Solution incubated with agitation in the dark at room temp for 2 hrs andovernight at 4′ C.1 ul of conjugate+9 ul water and 2 ul gel loadingbuffer (GLB) were loaded onto a 1% gel, as control 1 ul of aminocomboCwas loaded in a similar manner (see FIG. 2).

Crude Purification

25 ul of conjugate, 25 ul water and 10 ul GLB was loaded onto a 1% gel,run and the bands of 508/517 bp excised were glass wool purified and thevolume, 250 ul was made up to 500 ul with PBS and applied to a PD 10column pre-equilibrated with PBS and 7×500 ul fractions were collected.10 ul of each fraction was diluted 1:10 and added to a goat anti-mousecoated microtitre plate and incubated for 1 hr at room temp. Afterwashing 100 ul of 1:100 OliGreen was added and the plate read (see FIG.3).

PCR Amplification of Desalted Conjugate

A 16 ul aliquot of the conjugate diluted 1:1 k through to 1:10M in waterwas added to wells, together with 4 ul of PCR reaction mix and the PCRreaction undertaken (see FIG. 4).

Reaction Mix

10×55.0

MgCl 22.0

DNTP's 11.0

RevC 0.4

SsTemp 1.0

Water 18.0

hsTaq 2.8

Cycling

95 15:00/94 0:15 53 0:30 72 0:40 45 cycles/72 5:00

100 ul of PicoGreen was diluted in 1:200 in TE pH7.5 and added to thewells. After a 5 minute incubation the wells were read at 485/528 nm

MAQSSE—T4 DNA Polymerase & S1 Nuclease

Forward (Fwd) and reverse (Rev) oligonucleotide primers were designed toflank the MCS of a bacterial vector. These primers were designed toconsist of approximately 60% A+T and 40% G+C and exhibit no 3′ terminaldimer formation. A 750 bp insert containing no thermally significantannealing sites was cloned into the vector. Nested primers Fwdin andRevin were designed to amplify a 400 bp region within the insert, theseconsisted of approximately 50% A+T and 50% G+C and exhibited no 3′terminal dimer formation. PCR was optimised in terms of Tm gradient,cycle number and reaction mix for combinations of Fwd & Rev and Fwdin &Revin.

Single-Strand PCR Template (SSPT) synthesis was achieved in a two stageprocess.

(i) PCR of the vector DNA was undertaken with the Fwd and Rev primers,the product was visualised by agarose gel electrophoresis, excised andpurified. This was labeled QIA Fwd&Rev.

(ii) To produce a Rev primed SSPT, asymmetrical PCR was optimised interms of QIAFwd&Rev concentration, Fwd & Rev primer ratio and cyclenumber. Once produced the Rev primed single strand PCR product wasexcised from an agarose gel and purified.

SSPT Characterisation

To establish that Rev primed SSPT was a single strand an aliquot wasdiluted in a mix of 1× S1 buffer containing S1 nuclease. This wasincubated for 30 minutes at 37° C. a similar reaction using QIA Fwd&Revserved as negative control. These results were evaluated by agarose gelelectrophoresis.

To determine the optimal conditions for T4 DNA polymerase extension ofthe Fwd primer along the Rev primed SSPT, experiments were undertaken inwhich the serial dilutions of Rev primed SSPT were added to the T4 DNApolymerase mix and these were incubated over a range of test period.Following standard ethanol/salt precipitations and resuspension inwater, PCR was undertaken using the Fwdin & Revin primers. The resultswere evaluated visually using agarose gel electrophoresis. Once optimalconditions were established serial dilutions of Fwd were made and theprocedure repeated to demonstrate an association between diminishing Fwdprimer concentrations and signal intensity.

Fluorimetrical Quantitation

To quantify Fwd, serial dilutions were amplified using PCR after which a1:200 dilution of PicoGREEN in TE pH 7.8 was added. The fluorophore wasexcited at 485 nm and the emitted light read at 528 nm these values wereused to produce a standard calibration curve.

Immunoassay 1

The platform assay used to compare the sensitivity of the technique withcurrent labels was for human TSH (hTSH) using monoclonal antibodies fromMedix Biochemica and controls/calibrators from the Department ofClinical Chemistry at the Royal Liverpool University of Hospital.Typically tracer antibody was either labeled with biotin usingbiotinamidocaproate NHS ester (control) or amino modified Fwd using theheterobifunctional reagent sulfo SMCC. Capture antibody was diluted inbinding buffer and added to wells of 96 well polycarbonate plates andincubated overnight at room temperature. Following blocking the controlsand calibrator were added to the wells and incubated overnight at roomtemperature. Following washing, tracer antibody was added and incubatedat room temperature for four hours. In the case of the control assay,following washing, avidin D HrP was added to the wells, incubated for 30minutes. After washing TMB was added and the reaction stopped after 30minutes with concentrated acid. The plate was read at 450 nm. In thecase of the Fwd label, following washing the T4 DNA polymerase mixincluding Rev primed SSPT was added to the wells and incubated at 37° C.for 45 minutes. Following washing a mix containing S1 nuclease was addedand incubated at 37° C. and incubated for 30 minutes. Following a finalwash stage a PCR reaction mix including the Fwdin & Revin was added andPCR undertaken. An aliquot of dilute picoGREEN was then added to eachwell and the plate read using the fluorometer (results not shown).

Immunoassay 2

Wells from a 0.2 ml gamma irradiated Hybaid plate were coated with 150ul of 2 ug/ml 5405 mab in 50 mM NaHCO3 pH8.5, ON at RT. Wells wereblocked with 150 ul of 2% lactose, 0.2% BSA, 2 mM MOPS, pH7.0 for 1 hrat room temperature. 150 ul of serial diluted reconstituted BioRad3(containing hTSH) in PBS were added to wells and incubated overnight at4′ C. Following washing Biohit 9 washes PBS) 70 ul of dilute conjugatedesalt#2 in PBS was added to the wells and incubated at 4′ C. overnight.Following washing (Biohit 9 washes PBS, 9 washes water) 10 ul of PCRreaction mix was added to the wells and the PCR reaction undertaken

Reaction Mix

10×40.0

MgCl 16.0

dNTPs 8.0

revC 0.3

ss temp 0.8

water 332.9

hsTaq 2.0

95 15:00/94 00:15/53 00:30/72 00:40 45 cycles 72 5:00

100 ul of 1:200 PicoGreen was added and the plate read at 485/528(seeFIG. 5 and FIG. 6).

Multianalyte Methods

RevC was commercially synthesized containing a Rox fluorophore at its 5′terminal. PCR was undertaken under the following conditions ROX+ ROX−RevC 10X 10.0 10.0 10.0 MgCl 4.0 4.0 4.0 dNTP's 2.0 2.0 2.0 pBADCombo1:10 0.7 0.7 0.7 revCROX 1:10 0.7 0.7 revC 0.7 ssTemp 0.2 0.2 0.2 water81.9 81.9 81.9 hsTaq 0.5 0.0 0.5cycling 95 15:/ 94 :15/ 53 :30/ 72 :40 45 cycles/ 72 5:

1 ml of 1:200 dilution PicoGreen was added to each tube and thesolutions mixed before transfer to cyclindrical cuvettes. Solutions wereexcited at 475 nm and emission was monitored in the range 500-650 nmusing a spectrofluorimeter (See Rox.ppt)

10 ul of amino comboC diluted in water was added to wells followed by 10ul of reaction mix and PCR undertaken 10X 82.5 MgCl 33.0 DNTP's 10.0RevCrox 0.3 SsTemp 0.75 Water 283.1 HsTaq 3.095 15:00/ 94 0:15 53 0:30 72 0:40 45cycles/ 72 5:00

100 ul of 1:200 dilution of Picogreen in TE pH7.5 was added to thewells. After a 5 minute incubation the wells were read at 485/528 and485/620 nm (see FIG. 7).

MAQSSE—Palindromic Method

Forward (Fwd) and reverse (Rev) oligonucleotide primers were designed toflank the MCS of a bacterial vector. These primers were designed toconsist of approximately 60% A+T and 40% G+C and exhibit no 3′ terminaldimer formation. The Fwd and Rev primers were synthesized together witha combined Rev+Fwd primer (Combo). A 750 bp insert containing nothermally significant annealing sites was cloned into the vector. PCRwas optimised in terms of Tm gradient, cycle number and reaction mix forcombinations of Fwd & Rev, Combo & Rev.

Single-Strand PCR Template (SSPT) synthesis was achieved in a two stageprocess.

(i) PCR of the vector DNA with the Fwd and Rev primers was undertaken,the product was visualised by agarose gel electrophoresis, excised andpurified. This was labeled QIAFwd&Rev.

(ii) To produce a Rev primed SSPT, asymmetrical PCR was optimised interms of QIAFwd&Rev concentration, Fwd & Rev primer ratio and cyclenumber. Once produced the Rev primed single strand PCR product wasexcised from an agarose gel, purified and labeled, QIAssRev.

SSPT Characterisation

PCR was undertaken using QIAssRev and either Rev or Combo & Rev toconfirm that the SSPT would bind and correctly amplify in the presenceof Combo & Rev and not Rev in isolation. Serial dilutions of QIAssRevwere similarly amplified and visualised by agarose gel to demonstrate anassociation between diminishing QIAssRev and signal intensity.

Fluorimetrical Quantitation

To quantify QIAssRev, serial dilutions were amplified using PCR afterwhich a 1:200 dilution of PicoGREEN in TE pH 7.8 was added. Thefluorophore was excited at 485 nm and the emitted light read at 528 nmthese values were used to produce a standard calibration curve.

Comparison Immunoassay

The platform assay used to compare the sensitivity of the technique withcurrent labels was for human TSH (hTSH) using monoclonal antibodies fromMedix Biochemica and controls/calibrators from the Department ofClinical Chemistry at the Royal Liverpool University of Hospital.Typically tracer antibody was either labeled with biotin usingbiotinamidocaproate NHS ester (control) or amino modified Combo usingthe heterobifunctional reagent sulfo SMCC. Capture antibody was dilutedin binding buffer and added to wells of 96 well polycarbonate plates andincubated overnight at room temperature. Following blocking the controlsand calibrator were added to the wells and incubated overnight at roomtemperature. Following washing tracer antibody was added and incubatedat room temperature for four hours. In the case of the control assay,following washing, avidin D HrP was added to the wells, incubated for 30minutes. After washing TMB was added and the reaction stopped after 30minutes with concentrated acid. The plate was read at 450 nm. In thecase of the Combo label, following washing the PCR reaction mix wasadded and PCR undertaken. An aliquot of dilute picoGREEN was then addedto each well and the plate read using the fluorometer.

1-23. (canceled)
 24. An immuno polymerase chain reaction method ofdetecting at least one biological molecule in an assay sample, saidmethod comprising: i) providing a preparation comprising aligand:oligonucleotide conjugate and an assay sample, wherein saidligand binds said biological molecule to be detected in said assaysample; ii) adding a preparation comprising a single stranded templateDNA molecule which anneals to said oligonucleotide in (i),deoxynucleotide triphosphates, and a DNA polymerase that elongates theprimed oligonucleotide to produce a double stranded template DNAmolecule; and iii) amplifying said double stranded template DNA moleculewith a polymerase chain reaction which detects said template DNAmolecule using oligonucleotide primers adapted to amplify said doublestranded template DNA molecule.
 25. A method according to claim 24,wherein said method further comprises incubating the double strandedtemplate DNA molecule formed in (ii) with a nuclease which degrades thesingle stranded template DNA.
 26. A method according to claim 24,wherein said oligonucleotide primers adapted to amplify said doublestranded template DNA molecule comprise a palindromic sequence.
 27. Amethod according to claim 24, wherein said assay sample is selected fromthe group consisting of blood, serum, semen, lymph fluid, cerebrospinalfluid, tears, saliva, urine, and sweat.
 28. A method according to claim24, wherein said ligand is a polypeptide.
 29. A method according toclaim 28, wherein said polypeptide is an antibody or comprises at leastthe effective binding part of an antibody.
 30. A method according toclaim 29, wherein said antibody is a monoclonal antibody or comprisesthe Fab fragment of a monoclonal antibody.
 31. A method according toclaim 28, wherein said polypeptide is a receptor.
 32. A method accordingto claim 28, wherein said polypeptide is a ligand of a receptor.
 33. Amethod according to claim 32, wherein said polypeptide is thyroidstimulating hormone.
 34. A method according to claim 24, wherein saidbiological molecule is a polypeptide and is associated with a diseasecondition.
 35. A method according to claim 34, wherein said disease iscancer.
 36. A method according to claim 35, wherein said polypeptide isa tumor rejection antigen.
 37. A method according to claim 34, whereinsaid polypeptide is thyroid stimulating hormone.
 38. A method accordingto claim 28, wherein said polypeptide is a polypeptide expressed by apathogen.
 39. A method according to claim 38, wherein said polypeptideis expressed by a pathogen selected from the group consisting ofviruses, bacteria and parasites.
 40. A method according to any of claim24, wherein said method detects at least two biological molecules.
 41. Aligand:oligonucleotide conjugate comprising at least one biologicalmolecule bound to said ligand, which has annealed thereto a singlestranded template DNA by complementary base pairing between saidoligonucleotide and said template, wherein the annealed template iselongated by a DNA polymerase to form a double stranded DNA templatewhich is subsequently amplified by a polymerase chain reaction.
 42. Aligand:oligonucleotide conjugate according to claim 41, wherein saidligand is a polypeptide.
 43. A ligand:oligonucleotide conjugateaccording to claim 42, wherein said ligand is an antibody or comprisesat least the effective binding part of an antibody.
 44. A conjugateaccording to claim 43, wherein said antibody is a monoclonal antibody.45. A conjugate according to claim 41, wherein said biological moleculeis thyroid stimulating hormone.
 46. A method of detecting at least onebiological molecule in a sample to be tested, said method comprising: i)providing a preparation comprising; a) an assay sample to be tested; andb) a ligand:oligonucleotide conjugate; ii) incubating said preparationunder conditions which allow the binding of said conjugate to saidbiological molecule to form a complex; iii) contacting the complexformed in (ii) with a single stranded DNA template nucleic acid adaptedto anneal to a part of the oligonucleotide in said conjugate; iv)providing a DNA polymerase which is capable of elongating said singlestranded DNA template to form a double stranded DNA template andamplifying said double stranded DNA template in a polymerase chainreaction wherein oligonucleotide primers used in said amplificationcomprise a palindromic sequence.
 47. A method according to claim 46,wherein said method detects a plurality of biological molecules.